The goal of this project is to overcome limitations and high cost of current genome finishing methods. An integrated system, the UniTool(Tm), is proposed to be developed for fast and robust sequencing off BAC, YAC DNA by primer walking as well as for sequencing of homopolymer tracks in plasmid clones. Such system will be universally applicable for closing of hundreds of thousands of gaps, which have been generated during the "working draft" phase of the Human Genome Sequencing Project and which will be generated in "draft" phase of mouse and rat genome projects. During Phase I we will develop basic separate elements of the Unitool, which include, but not limited to, developing a cost effective dye terminator and dye-fimer BAC sequencing reactions, fimer design rules, selecting of "helper" DNA binding proteins, methods for problem regions, and developing of a processive DNA polymerase. During Phase II these separate solutions will be tested for compatibility and general applicability, bundled with a software for fimer design and will be ultimately integrated into a universal solution for genome finishing. PROPOSED COMMERCIAL APPLICATIONS: The UniTool system will be the first comprehensive, "crossplatform" solution to the problem of genome finishing. It will greatly increase the throughput of sequencing projects and drastically decrease their cost. UniTool will be compatible with automated operations at sequencing factories or service facilities and individual research by bench scientists.